A prospective cohort study of pain with intrauterine device insertion among women with and without vaginal deliveries.

The purpose of this prospective cohort study was to compare pain during IUD insertion between women with a history of vaginal delivery and women without a history of vaginal delivery. First-time IUD users chose either the CuT380A or the levonorgestrel IUS. We enrolled 49 women with previous vaginal delivery and 49 women with no history of vaginal delivery (either only caesarean deliveries or nulliparous). The mean pain score on a 0-100 mm visual analog scale during insertion in the vaginal delivery group was 34.7 (SD 31.6) compared with 51.2 (SD 29.2) in the group without previous vaginal delivery (p = 0.009). In multivariable analysis controlling for age, breast-feeding, expected pain, baseline anxiety, insertion timing (6-12 weeks postpartum, 2-4 weeks post-abortion or interval), and insertion difficulty, history of vaginal delivery was associated with a 15.5 point reduction in pain (95% CI, -27.4, -3.7). Other significant predictors of pain were 'expected pain' and 'insertion difficulty'.

Correlating birthweight with neurological severity of obstetric brachial plexus lesions.

OBJECTIVE: To investigate the nature and extent of neurosurgically treated obstetric plexus lesions with obstetric and neonatal precedents. DESIGN: Retrospective analysis of prospectively collected data. SETTING: Leiden, the Netherlands. POPULATION: A 9-year cohort of infants (n = 206) neurosurgically treated for obstetric brachial plexus lesion at a tertiary referral centre for nerve lesions. METHOD: Obstetric and neonatal data (parity, diabetic status, pregnancy gestation, mode of cephalic delivery and birthweight) were collected using a standardised protocol and correlated to neurological severity of the brachial plexus lesion. MAIN OUTCOME MEASURE: Neurological severity of the brachial plexus lesion. RESULTS: Nulliparous women delivered significantly lower birthweight newborns (P = 0.016), injuries in those infants were associated with the least severe injury classification. The most prominent association in ordinal logistic regression was between neurological injury severity and larger birthweight (P < 0.001). CONCLUSIONS: Birthweight is correlated with neurological severity of the injury in a group of infants experiencing brachial plexus injury resulting from cephalic vaginal delivery.

Human intrinsic factor secretion: immunocytochemical demonstration of membrane-associated vesicular transport in parietal cells.

The human gastric parietal cell synthesizes and secretes intrinsic factor (IF) and acid. In contrast to the cellular mechanisms of acid secretion, little is known about the mechanisms of IF secretion. To elucidate these mechanisms we obtained gastric secretions and sequential fundic biopsies from three subjects before and after pentagastrin stimulation (6 microgram/Kg s.c.). IF was localized in the biopsies using an ultrastructural immunoperoxidase technique using a well-characterized, monospecific antibody to human IF. IF output was quantified using a specific radioimmunoassay in concurrently obtained gastric secretions. Before stimulation, IF was associated with tubulovesicles scattered throughout the cytoplasm and with some in rough endoplasmic reticulum (RER). The tubulovesicles associated with IF migrated to the periphery of the secretory canaliculi within 8 min of stimulation. IF was present on secretory microvilli between 8 and 30 min when IF output in gastric juice was at its maximum. The cessation of IF secretion coincided with the depletion of IF associated with tubulovesicles. IF appeared in the perinuclear space and RER as the IF associated with tubulovesicles was secreted. These observations indicate that IF secretion depends upon membrane-associated vesicular transport and provides support for a membrane translocation-fusion hypothesis to explain the morphologic changes that occur in the parietal cell during secretion.

Immunocytochemical localization of the intrinsic factor-cobalamin receptor in dog-ileum: distribution of intracellular receptor during cell maturation.

Absorption of cobalamin is facilitated by the binding of the intrinsic factor-cobalamin complex (IF-cbl) to specific receptors in the ileum. The physical and biochemical characteristics of this ligand-receptor binding reaction have been extensively studied, but little is known about the cellular mechanisms or receptor synthesis, intracellular transport, and expression on the microvillus surface membrane. We attempted to delineate these mechanisms by using ultrastructural immunocytochemistry to localize the IF-cbl receptor in the crypt, mid-villus, and villus tip regions of mucosal biopsies obtained from the ileum of anesthetized dogs. Prior to initiating the ileal localization studies, the antisera to purified canine IF-cbl receptor that was employed in our studies was shown to have specificity for site (e.g., ileal enterocytes vs. other cells within the gastrointestinal tract) and immunohistochemical specificity. Receptor synthesis in endoplasmic reticulum begins in crypt enterocytes, but continues in cells throughout the villus. In the mid-villus region synthesized receptor translocates vectorially to the microvillus surface associated with membranous vesicles and then inserts into the microvillus pit. Receptor remains fixed to the microvillus pit and does not distribute uniformly over the brush border membrane. All villus tip enterocytes contained IF-cbl receptor in microvillus pits, vesicles, and endoplasmic reticulum, but in addition extensive perinuclear membrane staining was evident as well as re-internalized receptor associated with multivesicular bodies. Basolateral membranes contained no receptor at any level of the villus. These observations suggest that the IF-cbl receptor (a) translocates to the apical cell surface at the mid-villus region by transport in vesicles, (b) directly inserts into and then remains fixed in microvillus pits, (c) is elaborated on the luminal surface most extensively in villus tip cells, and (d) although reinternalized, does not move IF and/or cbl to the basolateral cell surface.

Apparent ruminal synthesis and intestinal disappearance of vitamin B12 and its analogs in dairy cows.

The aim of the project was to calculate the apparent synthesis or destruction of cobalamin (vitamin B(12)) and its analogs in the rumen as well as their apparent intestinal disappearance in dairy cows. Four lactating cows were fed a diet supplemented with cobalt alone (0.76 mg/kg of DM; control) or with cobalt and vitamin B(12) (cyanocobalamin, 500 mg/d; treated). In addition to cobalamin, the only biologically active molecule for the cow, 7 analogs were identified in duodenal and ileal digesta: cobinamide, which lacks the base, ribose, and phosphate groups; and 6 other molecules in which the base, 5,6-dimethylbenzimidazole, is replaced by cresol, 2-CH(3)-adenine, adenine, 2-CH(3)-S-adenine, or 5-OH-benzimidazole, or an unidentified cobamine. Small amounts of cobalamin and cobinamide were detected in the total mixed ration, but apparent synthesis of all forms took place in rumen. During the control period, cobalamin represented 38% of the total amounts of corrinoids produced in rumen. Approximately 11% of the average daily intake of cobalt was used for apparent ruminal synthesis of corrinoids, of which only 4% was incorporated into cobalamin. Only 20% of the supplement of cyanocobalamin was recovered at the duodenal level; cobinamide appeared to be the major product of degradation of supplementary cyanocobalamin in the rumen. During the control and treatment periods, there was an apparent intestinal disappearance of cobalamin and 5-OH-benzimidazole cobamide only; only the apparent intestinal disappearance of cobalamin differed between the 2 periods. Although cobalamin was not the major form synthesized by ruminal microflora and, even if supplementary cyanocobalamin was extensively destroyed by ruminal microflora, based on calculations of apparent intestinal disappearance, cobalamin seems to be the major form absorbed in the small intestine.

Subclinical vitamin B12 deficiency in pregnant women attending an antenatal clinic in Nigeria.

SUMMARY: Inadequate vitamin B12 status in a pregnant woman increases the risk for adverse maternal and fetal outcomes. The use of serum vitamin B12 concentration alone to assess vitamin B12 status in pregnant women is unreliable because of the decrease in serum vitamin B12 levels in normal pregnancy. The combination of serum vitamin B12 and methylmalonic acid (MMA) concentrations may provide a better estimate of vitamin B12 status. We obtained blood samples from 98 pregnant women in the third trimester at an antenatal clinic in Jos, Nigeria. All subjects were taking iron and folate supplements. Twelve of the subjects had a serum vitamin B12 concentration <148 pmol/l and 18 subjects had a serum MMA level >271 nmol/l. Using a combination of low serum vitamin B12 and elevated MMA concentrations, eight subjects were classified as having subclinical vitamin B12 deficiency. Because of the potential harmful consequences of vitamin B12 deficiency in pregnant women, it would be advisable to add vitamin B12 supplements to the existing regimen of folate and iron supplements currently provided to pregnant women in Nigeria.

Association between decreased vitamin levels and MTHFR, MTR and MTRR gene polymorphisms as determinants for elevated total homocysteine concentrations in pregnant women.

OBJECTIVES: To examine the association between methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C), methionine synthase (MTR) A2756G and methionine synthase reductase (MTRR) A66G gene polymorphisms and total homocysteine (tHcy), methylmalonic acid (MMA) and S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH) levels; and to evaluate the potential interactions with folate or cobalamin (Cbl) status. SUBJECTS/METHODS: Two hundred seventy-five healthy women at labor who delivered full-term normal babies. Cbl, folate, tHcy, MMA, SAM and SAH were measured in serum specimens. The genotypes for polymorphisms were determined by PCR-restriction fragment length polymorphism (RFLP). RESULTS: Serum folate, MTHFR 677T allele and MTR 2756AA genotypes were the predictors of tHcy levels in pregnant women. Serum Cbl and creatinine were the predictors of SAM/SAH ratio and MMA levels, respectively. The gene polymorphisms were not determinants for MMA levels and SAM/SAH ratios. Low levels of serum folate were associated with elevated tHcy in pregnant women, independently of the gene polymorphisms. In pregnant women carrying MTHFR 677T allele, or MTHFR 1298AA or MTRR 66AA genotypes, lower Cbl levels were associated with higher levels of tHcy. Lower SAM/SAH ratio was found in MTHFR 677CC or MTRR A2756AA genotypes carriers when Cbl levels were lower than 142 pmol/l. CONCLUSIONS: Serum folate and MTHFR C677T and MTR A2576G gene polymorphisms were the determinants for tHcy levels. The interaction between low levels of serum Cbl and MTHFR (C677T or A1298C) or MTRR A66G gene polymorphisms was associated with increased tHcy.

Markers of B-vitamin deficiency and frailty in older women.

OBJECTIVE: To evaluate the association between markers of vitamins B12, B6 and folate deficiency and the geriatric syndrome of frailty. DESIGN: Cross-sectional study of baseline measures from the combined Women's Health and Aging Studies. SETTING: Baltimore, Maryland. PARTICIPANTS: Seven hundred three community-dwelling women, aged 70-79. MEASUREMENTS: Frailty was defined by five-component screening criteria that include weight, grip strength, endurance, physical activity and walking speed measurements and modeled as binary and 3-level polytomous outcomes. Independent variables serum vitamin B6, vitamin B12, methylmalonic acid, total homocysteine, cystathionine and folate were modeled continuously and as abnormal versus normal. RESULTS: Serum biomarker levels varied significantly by race. All analyses were race-stratified and results are reported only for Caucasian women due to small African American sample size. In polytomous logistic regression models of 3-level frailty, Caucasian women with increasing MMA, defined either continuously or using a predefined threshold, had 40-60% greater odds of being prefrail (p-values < 0.07) and 1.66-2.33 times greater odds of being frail (p-values < 0.02) compared to nonfrails after adjustment for age, education, low serum carotenoids, alcohol intake, cardiovascular disease and renal impairment. Both binary and polytomous frailty models evaluating vitamin B12 as the main exposure estimated odds ratios that were similar in trend yet slightly less significant than the MMA results. CONCLUSIONS: These results suggest that vitamin B12 deficiency may contribute to the frailty syndrome in community-dwelling older women. Future studies are needed to explore these relationships longitudinally.

Absorption, plasma transport, and cellular retention of cobalamin analogues in the rabbit. Evidence for the existence of multiple mechanisms that prevent the absorption and tissue dissemination of naturally occurring cobalamin analogues.

Analogues of cobalamin (Cbl; vitamin B(12)) are prevalent in nature as a result of bacterial synthesis, and are of additional interest because of their potential use as antimetabolites and chemotherapeutic agents. We have synthesized 14 Cbl analogues containing (57)Co and have compared their gastrointestinal absorption, plasma transport, and cellular retention to that of [(58)Co]Cbl in rabbits. Many of the Cbl analogues were bound with low affinity by intrinsic factor, and none of these [(57)Co]Cbl analogues were taken up by the ileum or absorbed into the body in amounts comparable to that of [(58)Co]Cbl. The Cbl analogues that were bound by intrinsic factor with high affinity were taken up by the ileum but, in many cases, they were retained there in significant amounts. Most of the Cbl analogues were bound by plasma transcobalamin II with high affinity and all of these transcobalamin II-[(57)Co]Cbl analogue complexes were taken up by a variety of tissues in a manner that was indistinguishable from that of transcobalamin II-[(58)Co]Cbl. The few analogues that were bound by transcobalamin II with low affinity were taken up by tissues in lesser amounts, and 20-70% of these analogues was rapidly excreted in the urine as occurs with native Cbl when it is present in plasma in unbound form. All of the Cbl analogues were bound by the granulocyte R-type Cbl-binding protein with high affinity and all of the R-type protein-[(57)Co]Cbl analogue complexes were cleared rapidly from plasma exclusively by hepatocytes as occurs with R-type protein-[(58)Co]Cbl. Some Cbl analogues were released back into the plasma and were disseminated among a variety of tissues via transcobalamin II as occurs with native Cbl. Other Cbl analogues were retained in the liver and eventually excreted in the feces and urine without accumulating in other tissues. These studies indicate that intrinsic factor and the ileum prevent certain Cbl analogues from entering the body and that the granulocyte R-type protein and hepatocytes prevent the dissemination of certain Cbl analogues that may gain entry such as during infections with Cbl analogue-producing bacteria. The fact that transcobalamin II binds and transports a large number of Cbl analogues indicates that these protective mechanisms can be circumvented and supports the feasibility of using Cbl analogues as antimetabolites in vivo.

Binding and uptake of transcobalamin II by human fibroblasts.

We have used purified, (125)I-labeled human transcobalamin II (TC II), saturated with cobalamin (Cbl), to study the uptake process for the TC II-Cbl complex by intact normal cultured human skin fibroblasts. We have also investigated the possibility that a defect in one step of this process underlies that inborn error of Cbl metabolism-designated cbl C-in which mutant cells are unable to retain Cbl intracellularly or convert it to its coenzyme forms. TC II-Cbl binding at 4 degrees C reached a plateau after 3-4 hr; 95% of the bound (125)I was releasable with trypsin. Binding of TC II-Cbl at 4 degrees C could be inhibited by human and rabbit TC II-Cbl and human TC II devoid of Cbl but not by other Cbl-binding proteins, albumin, or free Cbl. Specific binding reached saturation at congruent with5 ng TC II/ml (0.13 nM) and could be inhibited by ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'- tetraacetic acid. At 37 degrees C, the TC II-Cbl complex was internalized as shown by a progressive decrease in the trypsin-releasable fraction of bound (125)I. After 2 h at 37 degrees C, increasing amounts of acid-soluble (125)I were found in the incubation medium indicating that the labeled TC II was being degraded. Chloroquine, an inhibitor of lysosomal proteolysis, prevented this degradation. The binding, internalization, and degradation of TC II-Cbl by cbl C cells was indistingusihable from that by control cells. Our studies provide additional support for the concepts: (a) that the TC II-Cbl complex binds to a specific cell surface receptor through a site on the TC II; (b) that the interaction between the receptor and TC II is calcium dependent; (c) that the TC II-Cbl is internalized via endocytosis; (d) that the degradation of TC II and release of Cbl from the complex occurs in lysosomes. We also conclude that the defect in cbl C must reside at some step beyond this receptor-mediated uptake process.

Isolation and characterization of an abnormal human intrinsic factor.

A patient has been described previously who presented at age 13 with vitamin B(12) (B(12)) deficiency secondary to a functionally abnormal intrinsic factor (IF). IF has now been isolated from the gastric juice of the patient, his sister, and both parents, who are first cousins, by using affinity chromatography on B(12)-Sepharose. Patient IF appeared normal in terms of (a) B(12) binding, (b) mol wt, (c) total amino acid and carbohydrate composition, and (d) immunodiffusion with rabbit anti-patient and anti-normal IF sera. After adsorption with normal IF, however, anti-patient IF serum precipitated the various IFs as follows: patient IF (> 95%); mother, father, and sister IF (50%); and normal IF (< 10%). Additional adsorption with mother, father, or sister IF completely inhibited the precipitation of patient IF. The association constant determined for patient IF-B(12) and human ileal mucosal homogenates (0.1 x 10(9) M(-1)) was 60-fold lower than that determined with normal IF-B(12) (6.0 x 10(9) M(-1)). Intermediate amounts of ileal IF-B(12) binding were observed with mother, father, and sister IF-B(12). These in vitro studies were supported by multiple Schilling tests, performed with a totally gastrectomized volunteer, that gave the following mean urinary excretions of [(57)Co]B(12): free B(12) (0.5%); + patient gastric juice (2.6%); + mother or father gastric juice (17%); and + normal gastric juice (26%). These studies demonstrate that the patient is homozygous and that the mother, father, and sister are heterozygous for a structurally abnormal IF that has a markedly decreased, but not absent, affinity for ileal IF-B(12) receptors. These studies also indicate that the B(12) and ileal binding sites are located on different portions of the IF molecule.

Inhibition of cobalamin-dependent enzymes by cobalamin analogues in rats.

To determine which parts of the cobalamin (cbl) molecule are required for enzyme activity and which parts, if altered, might inhibit cbl-dependent enzyme activity, we synthesized 16 cbl analogues and administered them to nutritionally normal rats. The cbl analogues, with either modifications of the propionamide side chains of the A-, B-, and C-rings, the acetamide side chain of the B-ring, or the nucleotide moiety, were administered to rats by continuous 14-d subcutaneous infusion. Infusion of cbl-stimulated, cbl-dependent activity. Changes in any part of the cbl molecule always abolished stimulation and, in some cases, caused potent inhibition of both cbl-dependent enzymes. The most inhibitory analogues, OH-cbl[c-lactam], a B-ring analogue, and OH-cbl[e-dimethylamide] and OH-cbl[e-methylamide], two C-ring analogues, decreased mean liver holo-L-methylmalonyl-coenzyme A mutase activity to 65% of control values and increased serum methylmalonic acid concentrations to as high as 3,200% of the control values. Liver methionine synthetase activity was decreased to approximately 20% of the control and mean serum total homocysteine concentrations were increased to 340% of control. A similar level of inhibition was demonstrated in rats who were exposed to 28 d of inhaled nitrous oxide or a prolonged period of dietary cbl deficiency. The inhibitory cbl analogues, nitrous oxide, and diet deficiency all depleted liver cbl. The naturally occurring cbl analogues with modifications of the nucleotide moiety had no effects. We conclude that all parts of the cbl molecule are necessary for in vivo cbl-dependent enzyme activity and that modifications of the side chains of the B and C rings are associated with potent in vivo inhibition of cbl-dependent enzyme activity.

The role and fate of rabbit and human transcobalamin II in the plasma transport of vitamin B12 in the rabbit.

Previous studies have shown that plasma transcobalamin II (TCII) facilitates the cellular uptake of [57Co] vitamin B12 (B12) by a variety of tissues, but the lack of an intrinsic label on the protein moiety of the TCII-B12 complex has made it impossible to determine the role and fate of TCII during this process. We have labeled homogensous rabbit and human TCII with 125I-labeled N-succinimidyl-3-(4-hydroxyphenyl) propionate and have performed in vivo experiments in rabbits. When 125I-labeled rabbit TCII-[57Co] B12 and 131I-labeled bovine albumin were simultaneously injected intravenously, we observed that 125Iand 57Co were cleared from plasma at a faster rate (t1/2 = 1 1/2 h) than 131I and that 125I and 57Co were present in excess of 131I in the kidney, liver, spleen, heart, lung, and small intestine 1/2 h after injection. Later, 57Co remained in excess of 131I, but the ratio of 125I to 131I decreased progressively in all of these plasma and were rapidly excreted in the urine. After 1 h following injection, 57Co was present in excess of 125I in the plasma...

Effect of proteolytic enzymes on the binding of cobalamin to R protein and intrinsic factor. In vitro evidence that a failure to partially degrade R protein is responsible for cobalamin malabsorption in pancreatic insufficiency.

Cobalamin (Cbl; vitamin B(12)) malabsorption in pancreatic insufficiency can be partially corrected by bicarbonate and completely corrected by pancreatic proteases but the mechanisms involved are unknown. Because saliva contains enough R-type Cbl-binding protein (R protein) to bind all of the dietary and biliary Cbl, it is possible that R protein acts as an inhibitor of Cbl absorption and that pancreatic proteases are required to alter R protein and prevent such inhibition. To test this hypothesis we studied the ability of R protein and intrinsic factor (IF) to compete for Cbl binding and ability of pancreatic proteases to alter this competition. Human salivary R protein bound Cbl with affinities that were 50- and 3-fold higher than those of human IF at pH 2 and 8, respectively. Cbl bound to IF was transferred to an equal amount of R protein with t((1/2))'s of 2 and 90 min at pH 2 and 8, respectively, and within several hours respective ratios of R protein-Cbl/IF-Cbl of 50 and 2 were observed. Cbl bound to R protein was not transferred to IF at either pH 2 or 8. Incubation of R protein with pancreatic proteases at pH 8 led to a 150-fold decrease in its affinity for Cbl. Incubation of R protein-Cbl with pancreatic proteases led to complete transfer of Cbl to IF within 10 min. Gel filtration studies with R protein-[(57)Co]Cbl and (125)I-R protein showed that pancreatic proteases partially degraded R protein. Pancreatic proteases differed in their ability to effect these changes with trypsin > chymotrypsin > elastase. Pancreatic proteases did not alter IF in any of the parameters mentioned above. Pepsin failed to alter either R protein or IF. THESE STUDIES SUGGEST THE FOLLOWING: (a) that Cbl is bound almost exclusively to R protein in the acid milieu of the stomach, rather than to IF as has been assumed previously; (b) that Cbl remains bound to R protein in the slightly alkaline environment of the intestine until pancreatic proteases partially degrade R protein and enable Cbl to become bound exclusively to IF; and (c) that the primary defect in Cbl absorption in pancreatic insufficiency is a lack of pancreatic proteases and a failure to alter R protein and effect the transfer of Cbl to IF. These studies also suggest that the partial correction of Cbl malabsorption observed with bicarbonate is due to neutralization of gastric HCl, since at slightly alkaline, pH IF can partially compete with R protein for the initial binding and retention of Cbl.

Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromatography-mass spectrometry.

To determine if levels of serum total homocysteine are elevated in patients with either cobalamin or folate deficiency, we utilized a new capillary gas chromatographic-mass spectrometric technique to measure total homocysteine in the serum of 78 patients with clinically confirmed cobalamin deficiency and 19 patients with clinically confirmed folate deficiency. Values ranged from 11 to 476 mumol/liter in the cobalamin-deficient patients and 77 of the 78 patients had values above the normal range of 7-22 mumol/liter as determined for 50 normal blood donors. In the cobalamin-deficient patients, serum total homocysteine was positively correlated with serum folate, mean corpuscular volume, serum lactate dehydrogenase, serum methylmalonic acid, and the degree of neurologic involvement, and inversely correlated with platelets and hematocrit. In the folate-deficient patients, values for serum total homocysteine ranged from 17 to 185 mumol/liter and 18 of the 19 patients had values above the normal range. Some patients with pernicious anemia who were intermittently treated with cyanocobalamin were found to have elevated serum levels of total homocysteine while they were free of hematologic and neurologic abnormalities. The measurement of serum total homocysteine will help define the incidence of cobalamin deficiency and folate deficiency in various patient populations.

Assay of methylmalonic acid in the serum of patients with cobalamin deficiency using capillary gas chromatography-mass spectrometry.

To determine the incidence of elevated levels of serum methylmalonic acid in patients with cobalamin deficiency, we utilized a new capillary gas chromatographic-mass spectrometric technique to measure methylmalonic acid in the serum of 73 patients with clinically confirmed cobalamin deficiency. Values ranged from 55 to 22,300 ng/ml, and 69 of the 73 patients had values above the normal range of 19-76 ng/ml as determined for 50 normal blood donors. In the cobalamin-deficient patients, serum methylmalonic acid was significantly correlated with the serum folate level and the degree of neurologic involvement. Some patients with pernicious anemia who were intermittently treated with cyanocobalamin were found to have elevated serum levels of methylmalonic acid while free of hematologic and neurologic abnormalities. A cobalamin-deficient patient is described with a normal serum cobalamin and an elevated serum methylmalonic acid. We conclude that the ability to measure methylmalonic acid in human serum will be useful in studies designed to determine the incidence of cobalamin deficiency in various patient populations.

Presence and formation of cobalamin analogues in multivitamin-mineral pills.

Because the origin of cobalamin (vitamin B12) analogues in animal chows and animal and human blood and tissues is unknown, we investigated the possibility that multivitamin interactions might convert cobalamin to cobalamin analogues. We homogenized three popular multivitamin-mineral pills in water, incubated them at 37 degrees C for 2 h, and isolated the cobalamin. Using paper chromatography we observed that 20-90% of the cobalamin was present as cobalamin analogues. Studies using CN-[57Co]cobalamin showed that these analogues were formed due to the concerted action of vitamin C, thiamine, and copper on CN-cobalamin. These cobalamin analogues are absorbed from the gastrointestinal tract of mice and either fail to stimulate or actually inhibit cobalamin-dependent enzymes when injected parenterally. We conclude that CN-cobalamin can be converted to potentially harmful cobalamin analogues by multivitamin-mineral interactions and that these interactions may be responsible for the presence of cobalamin analogues in animal chows and animal and human blood and tissues.

Isolation and characterization of a novel vitamin B12-binding protein associated with hepatocellular carcinoma.

High levels of a novel vitamin B12-binding protein (hepatoma B12 BP) have been observed recently in plasma obtained from three adolescent patients with hepatocellular carcinoma. This protein has now been isolated in homogeneous form from the plasma and pleural fluid of two of these patients by the use of affinity chromatography with vitamin B12-Sepharose. The hepatoma B12 BP belongs to the R-type group of B12-binding proteins and is essentially indistinguishable from the recently isolated human milk and saliva R-type proteins in terms of: (a) immunologic properties based on immunodiffusion and immunoprecipitation assays; (b) amino acid composition; (c) molecular weight based on amino acid and carbohydrate content; and (d) absorption spectra. Both hepatoma B12 BPs contain more sialic acid and less fucose than the milk and saliva B12 BPs. All four proteins contain similar amounts of galactose, mannose, galactosamine, and glucosamine. Differences in sialic acid content appear to account for the differences in electrophoretic mobility that were observed among the four proteins. Differences in total carbohydrate content appear to account for the differences in apparent molecular weight that were observed with both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor tissue from one of the patients contained 10 times as much R-type protein as did normal liver tissue from the same patient. This suggests, although it does not prove, that synthesis by the tumor is the cause of the high levels of R-type protein found in the plasma of certain patients with hepatocellular carcinoma. Plasma survival studies performed with rabbits indicate that the hepatoma B12 BP has a prolonged plasma survival and suggests that his parameter is also of importance.

Characterization of ileal vitamin B12 Binding using homogeneous human and hog intrinsic factors.

Elucidation of the mechanism of intrinsic factor (IF)-mediated vitamin B(12) (B(12)) binding to ileal binding sites has been hampered by the use of crude or only partially purified preparations of IF in previous studies. We have used homogeneous human IF and hog IF isolated by affinity chromatography to study [(57)Co]B(12) binding to ileal mucosal homogenates. The following observations were made: (a) Human IF-B(12) and hog IF-B(12) were bound to human, monkey, hog, dog, rabbit, mouse, hamster, and guinea pig ileal, but not jejunal, homogenates in amounts significantly greater than free B(12) or B(12) bound to five other homogeneous B(12)-binding proteins; (b) only IF-mediated B(12) binding was localized to ileal homogenates and was inhibited by EDTA; (c) values for the association constant (K(a)) for the various ileal homogenates mentioned above and human IF-B(12) and hog IF-B(12) ranged from 0.3 x 10(9) M(-1) to 13.0 x 10(9) M(-1). Apparent differences in the K(a) for human IF-B(12) and hog IF-B(12) existed in most species; (d) the number of ileal IF-B(12) binding sites per gram (wet weight) of ileal mucosa ranged from 0.3 x 10(12) to 4.9 x 10(12). The same value was always obtained with human IF-B(12) and hog IF-B(12) for any given homogenate preparation; (c) 100-fold excesses of free B(12) or human IF and hog IF devoid of B(12) did not significantly inhibit human IF-B(12) and hog IF-B(12) binding to human and hog ileal homogenates. THESE EXPERIMENTS PERFORMED WITH HOMOGENEOUS IF INDICATE THAT: (a) gastric factors other than IF are not required for B(12) binding to ileal IF-B(12)-binding sites: (b) the mechanism of ileal IF-B(12) binding is different from that of free B(12) or of B(12) bound to non-IF-B(12)-binding proteins; (c) human IF and hog IF have different structures; (d) human IF-B(12) and hog IF-B(12) bind to the same ileal binding sites; and (c) human and hog ileal IF-B(12) binding sites bind free B(12) and human and hog IF devoid of B(12) poorly, if at all.

Correction of cobalamin malabsorption in pancreatic insufficiency with a cobalamin analogue that binds with high affinity to R protein but not to intrinsic factor. In vivo evidence that a failure to partially degrade R protein is responsible for cobalamin malabsorption in pancreatic insufficiency.

In vitro studies indicate that [(57)Co]cobalamin (Cbl) is preferentially bound to salivary R protein as opposed to intrinsic factor (IF) and that [(57)Co]Cbl bound to R protein is not transferred to IF at either pH 2 or pH 8. Incubation of R protein-[(57)Co]Cbl with pancreatic proteases causes a partial degradation of the R protein moiety and a rapid transfer of [(57)Co]Cbl to IF. We have postulated that the etiology of Cbl malabsorption in pancreatic insufficiency is an inability to partially degrade R protein because of a lack of pancreatic proteases. We have tested this hypothesis by determining the ability of a nonradioactive Cbl analogue, bound with high affinity by R protein but not by IF, to correct the malabsorption of [(57)Co]Cbl in patients with pancreatic insufficiency.R protein bound the Cbl analogue known as cobinamide with affinities that were the same and only 14-fold lower than those for Cbl at pH 8 and pH 2, respectively. Cobinamide was bound by IF with affinities that were 600,000- and 10,000-fold lower than those for Cbl at pH 8 and 2, respectively. The addition of 125 pmol of nonradioactive cobinamide to 0.5 pmol of [(57)Co]Cbl before being added to 1 pmol of R protein and 1 pmol of IF, markedly inhibited the ability of R protein to compete with IF for binding the [(57)Co]Cbl. Similar results were obtained with freshly aspirated gastric juice. This change was essentially indistinguishable from that observed previously when R protein or R protein-[(57)Co]Cbl was incubated in vitro with trypsin. The oral administration of 100 nmol of nonradioactive cobinamide in Schilling tests was equivalent to trypsin in its ability to completely correct the malabsorption of 0.4 nmol of [(57)Co]Cbl in three patients with pancreatic insufficiency. The fact that both trypsin and nonradioactive cobinamide inhibit the ability of R protein to compete with IF for [(57)Co]Cbl binding in vitro, and correct the mal-absorption of [(57)Co]Cbl in patients with pancreatic insufficiency in vivo, supports our hypothesis that the primary defect in Cbl absorption in this disease is an inability to partially degrade R protein because of a lack of pancreatic proteases.